Research on fungal pathogens with the aim to identify virulence determinants strictly relies on the generation of defined, recombinant strains, a task that is executed by means of a sophisticated ...molecular biology toolbox. Recent developments in fungal genome engineering have opened a new frontier by implementing the CRISPR-Cas9 technology, based on expression of the Cas9 endonuclease that is loaded by a single guiding RNA (sgRNA) molecule to target a defined site in the recipient genome. This novel approach has been adapted successfully to engineer fungal genomes, among them the one of the human-pathogenic mould Aspergillus fumigatus Implementation of the required components was achieved by various means that differ with respect to expression of the Cas9 enzyme and sgRNA delivery. Validation of CRISPR-Cas9-mediated mutagenesis could be executed by targeting selected candidate genes of A. fumigatus to provide a promising perspective for screening and multiplexing approaches to scrutinize the virulome of this opportunistic fungal pathogen in a comprehensive manner, such as by analyzing genetic polymorphisms or the function of gene families.
Highlights • Aspergillosis is a manifold disease caused by the saprobe Aspergillus fumigatus. • A variety of cellular traits determines its interaction with the host. • Conidial constituents and cell ...wall components interfere with host immunity. • Secreted extracellular effectors shape the human virulome of A. fumigatus.
Sulphur is an essential element that all pathogens have to absorb from their surroundings in order to grow inside their infected host. Despite its importance, the relevance of sulphur assimilation in ...fungal virulence is largely unexplored. Here we report a role of the bZIP transcription factor MetR in sulphur assimilation and virulence of the human pathogen Aspergillus fumigatus. The MetR regulator is essential for growth on a variety of sulphur sources; remarkably, it is fundamental for assimilation of inorganic S-sources but dispensable for utilization of methionine. Accordingly, it strongly supports expression of genes directly related to inorganic sulphur assimilation but not of genes connected to methionine metabolism. On a broader scale, MetR orchestrates the comprehensive transcriptional adaptation to sulphur-starving conditions as demonstrated by digital gene expression analysis. Surprisingly, A. fumigatus is able to utilize volatile sulphur compounds produced by its methionine catabolism, a process that has not been described before and that is MetR-dependent. The A. fumigatus MetR transcriptional activator is important for virulence in both leukopenic mice and an alternative mini-host model of aspergillosis, as it was essential for the development of pulmonary aspergillosis and supported the systemic dissemination of the fungus. MetR action under sulphur-starving conditions is further required for proper iron regulation, which links regulation of sulphur metabolism to iron homeostasis and demonstrates an unprecedented regulatory crosstalk. Taken together, this study provides evidence that regulation of sulphur assimilation is not only crucial for A. fumigatus virulence but also affects the balance of iron in this prime opportunistic pathogen.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
The origin of isolates routinely used by the community of Aspergillus fumigatus researchers is periodically a matter of intense discussion at our centre, as the construction of recombinant ...isolates have sometimes followed convoluted routes, the documentation describing their lineages is fragmented, and the nomenclature is confusing. As an aide memoir, not least for our own benefit, we submit the following account and tabulated list of strains (Table 1) in an effort to collate all of the relevant information in a single, easily accessible document. To maximise the accuracy of this record we have consulted widely amongst the community of Medical Mycologists using these strains. All the strains described are currently available from one of these organisations, namely the Fungal Genetics Stock Centre (FGSC), FungiDB, Ensembl Fungi and The National Collection of Pathogenic Fungi (NCPF) at Public Health England. Display items from this manuscript are also featured on FungiDB.
Lay abstract
We present a concise overview on the definition, origin and unique genetic makeup of the Aspergillus fumigatus isolates routinely in use by the fungal research community, to aid researchers to describe past and new strains and the experimental differences observed more accurately.
The identification of cellular targets for antifungal compounds is a cornerstone for the development of novel antimycotics, for which a significant need exists due to increasing numbers of ...susceptible patients, emerging pathogens, and evolving resistance. For the human pathogenic mold
Aspergillus fumigatus
, the causative agent of the opportunistic disease aspergillosis, only a limited number of established targets and corresponding drugs are available. Among several targets that were postulated from a variety of experimental approaches, the conserved thioredoxin reductase (TrxR) activity encoded by the
trxR
gene was assessed in this study. Its essentiality could be confirmed following a conditional TetOFF promoter replacement strategy. Relevance of the
trxR
gene product for oxidative stress resistance was revealed and, most importantly, its requirement for full virulence of
A. fumigatu
s in two different models of infection resembling invasive aspergillosis. Our findings complement the idea of targeting the reductase component of the fungal thioredoxin system for antifungal therapy.
Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease caused by the fungus Aspergillus fumigatus, and is a leading cause of invasive fungal infection-related mortality and ...morbidity in patients with hematological malignancies and bone marrow transplants. We developed and tested a novel probe for noninvasive detection of A. fumigatus lung infection based on antibody-guided positron emission tomography and magnetic resonance (immunoPET/MR) imaging. Administration of a 64CuDOTA-labeled A. fumigatus-specific monoclonal antibody (mAb), JF5, to neutrophil-depleted A. fumigatus-infected mice allowed specific localization of lung infection when combined with PET. Optical imaging with a fluorochrome-labeled version of the mAb showed colocalization with invasive hyphae. The mAb-based newly developed PET tracer 64CuDOTA-JF5 distinguished IPA from bacterial lung infections and, in contrast to 18FFDG-PET, discriminated IPA from a general increase in metabolic activity associated with lung inflammation. To our knowledge, this is the first time that antibody-guided in vivo imaging has been used for noninvasive diagnosis of a fungal lung disease (IPA) of humans, an approach with enormous potential for diagnosis of infectious diseases and with potential for clinical translation.
Pathogen-pathogen interactions in polymicrobial infections are known to directly impact, often to worsen, disease outcomes. For example, co-infection with
and
, respectively the most common bacterial ...and fungal pathogens isolated from cystic fibrosis (CF) airways, leads to a worsened prognosis. Recent studies of
microbial cross-talk demonstrated that
-derived volatile sulfur compounds (VSCs) can promote
growth
. However, the mechanistic basis of such cross-talk and its physiological relevance during co-infection remains unknown. In this study we combine genetic approaches and GC-MS-mediated volatile analysis to show that
assimilates VSCs via cysteine (CysB)- or homocysteine (CysD)-synthase. This process is essential for utilization of VSCs as sulfur sources, since
-derived VSCs trigger growth of
wild-type, but not of a Δ
Δ
mutant, on sulfur-limiting media.
produces VSCs when infecting
and co-infection with
in this model results in a synergistic increase in mortality and of fungal and bacterial burdens. Interestingly, the increment in mortality is much greater with the
wild-type than with the Δ
Δ
mutant. Therefore,
ability to assimilate
derived VSCs significantly triggers a synergistic association that increases the pathobiology of infection. Finally, we show that
can promote fungal growth when growing on substrates that resemble the lung environment, which suggests that this volatile based synergism is likely to occur during co-infection of the human respiratory airways.
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